PROJECT SUMMARY Background: Arsenic (As) exposure through drinking water persists in many regions of the world. In Bangladesh, 40% of the population is exposed to As concentrations in drinking water exceeding the World Health Organization (WHO) standard of 10 ?g/L. Chronic As exposure affects numerous organs and systems, increasing the risk of health conditions including skin lesions, impaired intellectual function, cardiovascular disease, diabetes, inflammation, and cancers. The risk for adverse health effects associated with As exposure persists even decades after the exposure has ended. Changes in the epigenome, including DNA methylation (DNAm), may be a common mechanism underlying As-induced health outcomes. Micronutrients involved in methyl transfer (e.g., folate) and active DNA demethylation (e.g., vitamin C) may modify the association between As exposure and epigenetic dysregulation. Aims: The proposed research will utilize existing loci- specific DNAm data (5-methylcytosine, 5-mC; and 5-hydroxymethylcytocsine, 5-OHmC) from two existing studies of As-exposed Bangladeshi adults. The following aims will be addressed: (1) investigate the effects of As exposure on loci-specific %5-mC and %5-OHmC; (2) investigate effect modification of the association between As exposure and DNAm by folate and vitamin C status; and (3) identify biological and molecular functions associated with genes containing As-induced epigenetic dysregulation through gene ontology (GO) enrichment analyses. Aim 3 will include an exploratory component to develop a novel statistical approach to GO analyses to reduce bias due to uneven probe coverage among genes on DNAm microarrays. Significance and innovation: This research may provide insights to the epigenetic mechanisms underlying the association between chronic As exposure and disease, as well as differences in susceptibility due to nutritional status, which can inform strategies for minimizing health risks through therapeutic drugs or nutritional interventions. The proposed research is particularly innovative in addressing the relationship between As exposure, nutritional status, and DNAm (%5-mC and %5-OHmC); these associations have not previously been investigated on the loci-specific level. Investigating loci-specific DNAm through GO analyses will also allow for the identification of molecular and biological functions impacted by As-induced epigenetic dysregulation. This research addresses the NIEHS Strategic Plan goals including ?identify[ing] and understand[ing] fundamental shared mechanisms or common biological pathways, e.g.,?epigenetic changes?in order to enable the development of applicable prevention and intervention strategies? (NIEHS Goal 1) and ?understand[ing] individual susceptibility?resulting from environmental factors?to facilitate prevention and decrease public health burden? (NIEHS Goal 2).